In vivo animal model of human leukemia

ABSTRACT

The present invention provides a process for making an in vivo model of human leukemia. The process includes the steps of: pre-conditioning an immunodeficient rodent by administering to the rodent a sub-lethal dose of irradiation and injecting the rodent with an effective pre-conditioning amount of human fetal cord blood mononuclear cells; maintaining the rodent for from about 5 to 10 days; and injecting the rodent with an effective engrafting amount of primary human leukemia cells. An in vivo and in vitro model of human leukemia are also provided.

Funds used to support some of the studies reported herein were providedby the National Institutes of Health (NIH DK40218). The United StatesGovernment may, therefore, have certain rights in the inventiondisclosed herein.

TECHNICAL FIELD OF THE INVENTION

The field of this invention is leukemia. More particularly, thisinvention pertains to models of human leukemia including an in vivorodent model of human leukemia.

BACKGROUND OF THE INVENTION

T-cell acute lymphoblastic leukemia (T-ALL) comprises −20% of ALL(Kersey J H., Blood 1997; 90: 4243-4251), with ALL being the most commontype of cancer in children. A better understanding of the biology ofT-ALL at the molecular level would facilitate the development ofselective therapy that exploits specific biological properties of theleukemia, thereby improving the outlook for this disease. Even though anumber of leukemia cell lines of T-cell origin have been establishedfrom patients (Gjerset R, et al., Cancer Res 1990; 50: 10-14; Smith S D,et al., Blood 1978; 52: 712-718; Lange B, et al., Blood 1987; 70:192-199; and Smith S D, et al., Cancer Res 1984; 44: 5657-5660),difficulty in maintaining primary cultures of leukemia cells frompatients has impeded study of the development of the disease.

Leukemic progenitor cells have been implicated in the maintenance andexpansion of leukemic blast populations (Uckun F M, et al., Immunology1988; 140: 2103-2111; Uckun F M, et al., Blood 1990; 76: 1723-1733).These clonogenic blast cells comprise only 0.05 to 1.5% of the bulkmarrow or peripheral blood blasts from ALL patients (Uckun F M, et al.,Immunology 1988; 140: 2103-2111; Touw I, et al., Blood 1986; 68:1088-1094), identified on the basis of their ability to proliferate andform colonies in semi-solid media in response to specific growth factors(Touw I, et al., Blood 1986; 68: 1088-1094; Touw I, et al., Blood 1985;66: 556-561). It is generally assumed that the colony-forming blastsrepresent the in vitro counterparts of the in vivo ALL blast progenitors(Uckun F M, et al., Immunology 1988; 140: 2103-2111). Despite these invitro studies, leukemia-initiating cells were not demonstrated in vivountil recently (Holyoake T, et al., Blood 1999; 94: 2056-2064; Ailles LE, et al., Nat Med 1997; 3: 730-737; and Terpstra W, et al., Blood 1996;87: 2187-2194).

The ability to engraft T-ALL cells directly from patient samples intoimmunodeficient rodents such as Nonobese Diabetic×Severe CombinedImmunodeficient (NOD/scid) mice would be uniquely valuable in thisregard, as well as for predicting the clinical course of the disease,detecting residual disease, and developing individualized therapeuticstrategies. The availability of a robust in vivo mouse model for T-ALLwould expedite characterization of the corresponding leukemia-initiatingcell, as well as delineation of cellular hierarchy within the leukemia.Pre-conditioning sub-lethally irradiated immunodeficient NOD/scid micewith human cord blood mononuclear cells (MNCs) facilitates thesubsequent engraftment in these mice of primary T-ALL cells obtainedfrom patients at the time of diagnosis. The present invention provides,in great detail a novel in vivo model of human leukemia engraftment. Thedata show that the level of engraftment depends on both the number ofcord blood MNCs and T-ALL cells injected. In addition, the data documentthe fidelity of the model to the human pathology with regard to thepattern of leukemia dissemination, as well as with regard to themaintenance of the leukemia-initiating cell within theleukemia-engrafted mouse. The data also provide evidence that the cordblood pre-conditioned NOD/scid mouse is applicable to the study of otherhuman leukemias in addition to T-ALL.

BRIEF SUMMARY OF THE INVENTION

The present invention provides a process for making an in vivo model ofhuman leukemia. The process includes the steps of: pre-conditioning animmunodeficient rodent by administering to the rodent a sub-lethal doseof irradiation and injecting the rodent with an effectivepre-conditioning amount of mononuclear cells (MNCs) derived from humanfetal cord blood; maintaining the rodent for 3 to 12 days, preferablyfrom about 5 to 10 days and, more preferably from about 6 to 9 days; andinjecting the rodent with an effective engrafting amount of primaryhuman leukemia cells. In one embodiment, the cord blood MNCs are stromalcells that comprise mesenchymal stem cells.

The immunodeficient rodent is preferably an immunodeficient mouse, morepreferably a NOD/scid mouse. Pre-conditioning the mouse includes twosteps. First, the mouse is irradiated with a sub-lethal dose of gammaradiation. The irradiation is whole body irradiation. A preferredsub-lethal radiation dose is from about 200 to about 500 rads. Morepreferably, the dose is from about 300 to about 400 rads and, even morepreferably about 350 rads.

Immediately following irradiation, the mouse is injected withmononuclear cells from fetal cord blood from a normal human subject. Apreferred effective number of mononuclear cells is from about 10⁶ toabout 10⁸ cells. More preferably, about 10-25×10⁶ cells are injected.About 1 week after pre-conditioning, the mouse is injected with viableprimary human leukemia cells. A preferred number of primary leukemiacells is from about 10⁶ to about 10⁷ cells. An especially preferrednumber of primary leukemia cells is 1-5×10⁶ cells.

In a related aspect, an in vivo model of human leukemia can be producedas set forth above using stromal cells derived from bone marrow. Thestromal cells of the bone marrow comprise stem cells of mesenchymalnature. A process of this invention can use any stem cells, especiallymesenchymal stem cells, as the pre-conditioning agent. The presentinvention also provides in vivo models of human leukemia produced by aprocess of this invention.

In another aspect, the present invention provides an immunodeficientrodent having engrafted human leukemia cells. Preferably, theimmunodeficient rodent is an immunodeficient mouse, more preferably isan NOD/scid mouse. The mouse is irradiated, injected with mononuclearcells derived from human fetal cord blood and then injected with humanprimary leukemia cells. In another aspect of the invention theirradiated mouse is injected with mesenchymal stem cells derived fromcord blood or bone marrow and then injected with human primary leukemiacells. The engrafted leukemia cells are found in the bone marrow andspleen of the mouse.

The efficient engraftment and subsequent expansion of the leukemiawithin pre-conditioned rodents affords a viable window for addressing atthe molecular level all events up to and including the expansion. Thedissemination of engrafted primary leukemic cells within thepre-conditioned rodent mimics the findings for the human pathology. Thelevel of primary cell engraftment increases both with increasing numberof pre-conditioning cells (e.g., MNCs, stem cells) and with increasingnumber of primary leukemia cells injected.

In a related embodiment, the present invention provides a method ofscreening anti-cancer drugs. In particular, the screening method can beused to screen anti-leukemia drugs or agents. The method includes thestep of administering to the animal model of this invention a putativeanti-leukemic agent and monitoring the effects of the drug on the courseof leukemia in the model.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings that form a portion of the specification FIG. 1 shows aprotocol for cord blood pre-conditioning of NOD/scid mice and analysisof the engraftment of primary human leukemia.

FIG. 2 shows leukemia-engrafted bone marrow and spleen from a cord bloodpre-conditioned mouse injected with primary T-ALL cells. Mice wereanalyzed by flow cytometry for T-ALL engraftment in bone marrow (A) andspleen (B). For each panel, the filled histogram curve corresponds tothe indicated experimental monoclonal antibody (mAb) and is superimposedover an open histogram corresponding to the istotype control mAb. Thefraction of cells staining positive for the experimental mAb wasdetermined by subtraction of the curves, using CellQuest 3.2.1 software.The percentage of human CD45⁺, CD7⁺, and CD19⁺ cells is indicated in thepanels.

FIG. 3 shows engraftment of primary T-ALL in mouse bone marrow for aseries of primary T-ALL donors. The level of T-ALL engraftment wasdetermined by flow cytometry, on the basis of CD45, CD7, and CD5expression.

FIG. 4 shows that the level of engraftment in mouse bone marrow andspleen is dependent on both the number of cord blood MNCs and the numberof T-ALL cells. In (A), mice were injected with the indicated number ofcord blood MNCs 7 days prior to the injection of 2.5×10⁶ primary T-ALLcells; the mice were sacrificed for analysis 6 weeks after injection ofthe T-ALL cells. In (B), mice were injected with 10×10⁶ cord blood MNCs7 days prior to the injection of the indicated number of primary T-ALLcells; the mice were sacrificed for analysis 6 weeks after injection ofthe T-ALL cells. The percentage of T-ALL in engrafted bone marrow(□-□-□) and spleen (●●-●) was determined flow cytometrically as CD7⁺CD5⁺ cells, the phenotype of the primary T-ALL in each case.

FIG. 5 shows the maintenance of the T-ALL leukemia initiating cellwithin the leukemia-engrafted mouse. In (A), the first mouse wasinjected with primary T-ALL cells obtained from a patient. In (B),however, the second mouse was injected with T-ALL cells in engraftedspleen recovered from the mouse in (A). Specifically, in (A) mice wereinjected with 10×10⁶ cord blood MNCs 9 days prior to the injection of1.6×10 ⁶ primary T-ALL cells; the mouse was sacrificed for analysis 7weeks after injection of the T-ALL cells. In (B) mice were injected with25×10⁶ cord blood MNCs 8 days prior to the injection of the indicatednumber of T-ALL cells obtained from engrafted spleen recovered from themouse in (A); the mouse was sacrificed for analysis 5 weeks afterinjection of the T-ALL cells. The percentage of T-ALL in engrafted bonemarrow was determined flow cytometrically as CD7⁺CD5⁺ and CD7⁺Vβ2⁺cells. The CD7 and TCR Vβ2 profiles are presented here.

FIG. 6 shows the engraftment of primary childhood B-ALL in cord bloodpre-conditioned mice. Specifically, mice were injected with 25×10⁶ cordblood MNCs 9 days prior to injection with 5×10⁶ primary B-ALL cells. Themice were sacrificed for analysis 6 weeks after injection of the B-ALLcells. The percentage of engrafted B-ALL in bone marrow (A) and spleen(B) was determined flow cytometrically as CD45⁺CD 19⁺ cells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides models of leukemia including an in vivoanimal model of human leukemia. A preferred animal for use with the invivo model is a rodent and, more particularly, a mouse. The rodent isimmunodeficient. That is, the animal lacks the normal capacity torespond to an insult with an immunological response. Numerousimmunodeficient rodent models are well known in the art. An especiallypreferred immunodeficient animal is a severe combined immunodeficientmouse (scid mouse). Means for obtaining such scid mice are well known inthe art. An especially preferred scid mouse for use in the presentinvention is a Nonobese Diabetic×severe combined immunodeficient(NOD/scid) mouse.

The immunodeficient animal contains engrafted human leukemia cells. Asused herein, the term “engrafted” and its grammatical equivalents meanstransplanted cells that have migrated throughout the organism toparticular tissues. Engrafted leukemia cells can be found throughout theanimal model. More particularly, engrafted leukemia cells are found inthe liver (portal and mesenchymal areas), kidney (perivascular andperiglomerular interstitial spaces), lung (parenchymal tissue), thymus,adrenal gland and peripheral blood. The largest number of engraftedcells is found in the hemopoietic tissues, bone marrow and spleen.

An in vivo model of leukemia according to the present invention has avariety of uses. One, the model can be used to study leukemogenesis.Two, the model can serve as a vehicle for testing the efficacy ofvarious treatments. Third, the model can be used as a vehicle forscreening putative antileukemia therapeutic agents. Fourth, the modelcan serve as a mean for continuous expansion of patient's leukemia cellsfor diagnosis/research purposes.

A detailed description of how to make an in vivo animal model of humanleukemia is set forth hereinafter below. NOD/scid mice (Shultz L D, etal., J. Immunol 1995; 154: 180-191) were bred and maintained in aspecific pathogen-free environment at The Scripps Research Institutevivarium in sterile Micro-Isolator cages and ventilated mouse racks (LabProducts, Seaford, Del.) without antibiotics. Five to six week old miceof either sex (but matched within a given experiment) were used in thepresent study.

Heparinized peripheral blood or bone marrow samples were obtained frompatients with childhood T-ALL who enrolled in protocol #9400 PediatricOncology Group. Analogous samples were also obtained from patients withchildhood B-cell acute lymphoblastic leukemia (B-ALL) or acutemyeloblastic leukemia (AML). In one study, the mononuclear cell (MNC)fraction from peripheral blood/bone marrow was isolated by Ficoll-Paquedensity gradient separation (Pharmacia, Piscataway, N.J.). The contentof lymphoblasts, as determined by Wright stain, was generally >90%. Insome cases, MNCs of leukemia samples were cryopreserved and stored inliquid nitrogen before use in the studies. Viability on thawing wasgenerally greater than 80% as determined by trypan blue dye exclusion.

Fetal cord blood samples were obtained from umbilical cord scheduled fordiscard, according to procedures approved by our Institutional ReviewBoard. After Ficoll-Paque density gradient centrifugation, the MNCs werecollected and washed with RPMI 1640 medium containing 2% fetal calfserum (FCS). Cord blood was used for injection as described below.

In a second study the umbilical cord MNCs were placed in culture, wherean adherent layer of cells containing mesenchymal stem cells wasobserved. Human cord blood MNCs were seeded at 1.5×10⁶ cells/ml in 10%fetal calf serumi/RPMI 1640 medium and cultured for two weeks withweekly change of medium. Certain of the cells adhered to the cultureplates. These adherent cells were evaluated and found to containmesenchymal stem cells capable of differentiating into various cells,including osteoblasts and adipocytes. These mesenchymal stem cells wereisolated and used in pre-conditioning as set forth below.

The protocol for pre-conditioning of NOD/scid mice and analysis of theengraftment of primary human leukemia is outlined in FIG. 1. Prior toleukemia implantation, the mice received 350 rads total body irradiationfrom a ¹³⁷CS γ-irradiator. Immediately thereafter, 10-25×10⁶ cells wereinjected in 0.25 ml sterile PBS via tail vein. Six to nine days later,1-5×10⁶ viable primary leukemia cells from a patient were suspended in0.25 ml PBS and injected via tail vein. For a given experiment, leukemiacells from a single donor were used for all mice. Experimental mice weretypically set up as two to four replicates. Mice were sacrificed whenthey became moribund with disseminated leukemia or electively between 5and 7 weeks after the leukemia cell injection. Necropsies wereperformed, and the burden of leukemia cells in mouse tissues wasdetermined by flow cytometry and histocytochemistry as described below.

Gross examination of various mouse tissues was performed afterlaparatomy immediately after sacrifice. Multiple tissues from mice(including liver, kidney, lung, and brain) were fixed in aqueousbuffered zinc formalin (Z-fix; Anatech, Battle Creek, Mich.),dehydrated, and embedded in paraffin by routine methods. Glass slideswith 4 μm tissue sections were prepared and stained withhematoxylin/eosin. The bone marrow of mice was collected from femurs andtibias. A single cell suspension was prepared by gentle pipetting.Spleen cells were collected by gentle dissociation. Red blood cellswithin the bone marrow and spleen cell suspensions were lysed usingbuffered ammonium chloride. Cell debris was removed by filtrationthrough a sterile nylon cell strainer (Becton Dickinson, San Jose,Calif.).

Multi-parameter analysis of single-cell suspensions from mouse bonemarrow and spleen was carried out using a FACScan flow cytometer (BectonDickinson). Two-color immunofluorescence was used to identify humanleukemia cells. Fluorescein isothiocyanate (FITC)- or phycoerythrin(PE)-conjugated mouse anti-human monoclonal antibodies (mAbs) wereobtained from PharMingen (San Diego, Calif.), with the exception ofPE-conjugated anti-TCR V β2 (clone MPB2D5, Coulter, Miami, Fla.). ThemAbs used in the work presented here include those directed againsthuman CD5 (clone UCHT2), CD7 (M-T701), CD 19 (HIB 19), CD33 (WM53), andCD45 (HI30). During analysis, red blood cells and debris were gated outon the basis of forward angle and 90° side scatter. At least 15,000events were collected for each sample. Istoype-matched control mAbs[FITC- or PE-conjugated IgG1 (clone MOPCO21)] were used to determine theappropriate cursor settings for analysis. Using CellQuest 3.2.1 software(Becton Dickinson), data were analyzed and displayed by two-dimensionalplots and by one-dimensional histograms.

Pre-conditioning sub-lethally irradiated immunodeficient NOD/scid micewith human cord blood mononuclear cells (MNCs) facilitates thesubsequent engraftment in these mice of primary leukemia cells obtainedfrom patients with T-ALL. As outlined in FIG. 1, in this modelirradiated NOD/scid mice are injected with human cord blood MNCsapproximately 1 week prior to the injection of primary leukemia cellsobtained from patients. The mice are then sacrificed approximately 6weeks later and the level of leukemia cell engraftment determined. Atypical profile of T-ALL engrafted mouse bone marrow and spleen, asassessed by flow cytometry, is presented in FIG. 2. CD45 expression isindicative of total human hematopoietic cell engraftment. CD7 isexpressed by engrafted human T-ALL. CD 19 is indicative of engraftedhuman cells of the B cell lineage. For this experiment, CD45⁺CD7⁺engrafted T-ALL cells comprise approximately 83% of bone marrow and 68%of spleen, as indicated by the corresponding histograms presented inFIG. 2. Notably, there are very few CD 19⁺ cells (approximately 2% inbone marrow and 4% in spleen) in the T-ALL engrafted mouse, suggestingthat expansion of the T-ALL overtakes the expansion of normal CD 19⁺cells developing from engrafted cord blood MNCs (Vormoor J, et al.,Blood 1994; 83: 2489-2497; Hogan C J, et al., Blood 1997; 90: 85-96;Kollmann T R, et al., Immunology 1994; 91: 8032-8036; Yu J., I FormosMed Assoc 1996; 95: 281-293; and Pflumio F, et al., Blood 1996; 88:3731-3740). In some cases, further confirmation that the engrafted cellswere derived from the injected primary T-ALL was carried out by analysisof TCR Vβ gene usage. Similar results were obtained from studies usingthe mesenchymal stem cells, which were shown to enhance the engraftmentof human leukemia cells.

Current studies also characterized the level of T-ALL engraftment inmouse bone marrow for a series of primary T-ALL donors over a range ofinjected T-ALL cell number (FIG. 3). In these studies, eight differentprimary T-ALL donors were used. Efficient engraftment in mouse bonemarrow typically is observed at 6 weeks following injection of 1-5×10⁶primary T-ALL cells into a mouse which has been pre-conditioned withcord blood (FIG. 3). Studies then addressed the issue of whether thelevel of engraftment in mouse bone marrow and spleen at 6 weeks isdependent on the number of cord blood MNCs and the number of primaryT-ALL cells injected (FIG. 4). In FIG. 4, two different experiments wereset up, with the same primary T-ALL donor but different cord blooddonors. From FIG. 4A it is apparent that the level of T-ALL engraftmentin mouse bone marrow and spleen at 6 weeks is dependent on the number ofcord blood cells used for pre-conditioning. Analogously, from FIG. 4B itis apparent that the level of T-ALL engraftment in bone marrow andspleen at 6 weeks is dependent on the number of primary T-ALL cellsinjected.

In order to address the likely utility of the present mouse model forthe study of T-ALL metastasis and the corresponding therapeuticintervention, the profile of T-ALL dissemination was determined in theengrafted, cord blood pre-conditioned mouse. In liver there were notableinfiltrations of leukemia cells in portal and mesenchymal areas. Inkidney, human leukemia cells are aggregated in perivascular andperiglomerular interstitial spaces. In the lung, leukemia cells weredetected within the parenchymal tissue. Engrafted T-ALL cells alsodisseminated to mouse thymus, adrenal gland, and peripheral blood.

Because of its central role in leukemia formation, it was ofconsiderable interest to determine whether in our model system theleukemia-initiating cell was maintained within the leukemia-engraftedmouse and was not, for example, exhausted in the course of T-ALLexpansion in vivo. To address this question, it was determined whetherT-ALL recovered from the engrafted spleen of a mouse injected withprimary T-ALL obtained from a patient could recapitulate the leukemia ontransfer to a second, cord blood pre-conditioned mouse. For this work, aprimary T-ALL that expresses T cell receptor chain variable region 2(TCR Vβ2) was used. In this way, the engrafted T-ALL could be uniquelyidentified as CD7⁺Vβ2⁺ cells. The results of this experiment arepresented in FIG. 5 for mouse bone marrow.

Injection of primary T-ALL cells obtained from the patient intopre-conditioned mice led to high-level engraftment in bone marrow,determined here at 7 weeks after the T-ALL injection (FIG. 5A).Specifically, 92% of the cells in bone marrow expressed CD7 and 89%expressed Vβ2, consistent with the existence of a CD7⁺Vβ2⁺ subsetcomprising approximately 90% of the bone marrow cells. In the samemouse, CD7⁺Vβ2⁺ cells (T-ALL) accounted for approximately 92% of spleencells. The T-ALL cells in engrafted spleen were used to inject a secondmouse that had been pre-conditioned with cord blood (FIG. 5B). Analysisof this second mouse recipient at 5 weeks after T-ALL injectionindicated recapitulation of the leukemia, with 58% of the bone marrowcells expressing CD7 and 52% expressing TCR Vβ2 (consistent withapproximately 52% of bone marrow cells having the CD7⁺Vβ2⁺ phenotype).In this second mouse recipient, the level of T-ALL engraftment in spleen(CD7⁺Vβ2+ cells) was 68%. These results indicate unambiguously thatthere is maintenance of the leukemia-initiating cell within theleukemia-engrafted mouse.

Although the model system was developed initially to facilitate study ofT-ALL and the pre-clinical testing of associated therapeutic strategies,it was of interest to determine whether it could be applied to the invivo study of other leukemias. To this end, cord blood pre-conditionedmice were injected with primary childhood B-cell acute lymphoblastoidleukemia (B-ALL) cells and the level of B-ALL engraftment in mouse bonemarrow and spleen determined on day 39 after B-ALL injection (FIG. 6).Approximately 90% of mouse bone marrow cells expressed a uniformCD45⁺CD19⁺ human phenotype expected for the engrafted B-ALL. Moreover,46% of spleen cells expressed the identical CD45⁺CD19⁺ phenotype (FIG.6B). Preliminary work suggests that the cord blood pre-conditioned mousemay also be applicable to the in vivo study of acute myeloblasticleukemia (AML). In this work, 16% leukemia engraftment in bone marrowand 1% engraftment in spleen were observed for a preconditioned NOD/scidmouse injected 11 days previously with AML.

Enhancement of Leukemia Colony Formation by Cord Blood or MesenchymalStem Cell Conditioned Medium In Vitro

To characterize the nature of the enhancing activity on leukemiaengraftment, colony formation of leukemia clonogenic cells was examinedin vitro in the presence of cord blood or cord blood derived mesenchymalstem cell conditioned medium. The in vitro leukemia colony assay isbased on the ability of leukemic clonogenic cells to proliferate andform colonies in response to growth factors such as IL-2. These leukemic“progenitor” cells have been implicated in the maintenance and expansionof leukemic blast populations.

The leukemia colony formation of primary T-ALL obtained from patientsand cultured with 100 units/ml of IL-2 and 10 ng/ml of phorbol12-myristate 13-acetate (PMA) in methylcellulose was significantlyenhanced by the addition of MNC or stem cell conditioned medium, in adose-dependent manner. The enhancement of T-ALL colony formation is asgreat as 4-fold by factor(s) present in the conditioned medium wassubstantially increased compared to that observed in the absence ofconditioned medium. On microscopic analysis, there is a wide range ofcolony sizes in the samples to which conditioned medium was added. Thecolonies in the cultures were individually picked, pooled for similarsizes of colonies, and the number of leukemia cells counted. It wasfound that the number of cells per individual colonies in cultures withcord blood conditioned medium ranged from 250 and 285×10²/colony, ascompared to less than 100 cells/colony observed in the absence ofconditioned medium. Therefore, this increase in burst size due to theaddition of cord blood conditioned medium in the cultures was in theorder of several fold to more than 100-fold.

To further confirm that cord blood constitutively expressed in vitro afactor(s), which enhances in vitro leukemia colony formation, a doublelayer agar assay was performed for leukemia samples. Some “diffusablefactor(s)” secreted from irradiated cord blood in the lower agar layersignificantly promoted plating efficiency of leukemia colonies in theupper layer in the absence of exogenous IL-2 and PMA. As would beexpected, the number of leukemia colonies on day 14 depends both on thenumber of irradiated cord blood MNCs in the lower agar layer as well ason the number of input T-ALL cells in the upper agar layer. The absenceof colonies when only the irradiated cord blood is cultured indicatesthat the colonies in the upper layer are derived from the T-ALLpreparations.

These in vitro studies of leukemia colony formation show thatproliferation of leukemic cells was likely stimulated by the addition ofMNC or stem cell conditioned medium. To confirm that the cells recoveredfrom individual colonies in the assay were primarily of leukemic originand not simply normal T cells from the patient's blood, we tookadvantage of an atypical surface phenotype of one patient's primaryT-ALL. Phenotypic analysis of the primary T-ALL leukemia sample obtainedfrom this patient revealed that these cells are CD7⁺ (99%), CD34⁺ (98%),CD45⁺ (2%) and negative for B cell and monocyte markers. Although theseleukemic cells expressed CD7 as expected, they anomalously failed toexpress CD45 and uniformally expressed CD34. This aberrant phenotypethus permits unambiguous discrimination of the patient's T-ALL cellsfrom normal T cells.

Using this primary leukemia sample, leukemia colony forming assay withthe addition of cord blood conditioned medium was performed. It wasshown that the addition of cord blood conditioned medium greatlyenhanced leukemia colony formation of MNCs from this patient by morethan three fold as expected. On day 14, leukemia colonies wereindividually picked. Approximately 1.4×10⁶ cells were recovered, andthese cells were pooled for flow cytometric analysis. The vast majorityof cells are uniquely CD45-CD7⁺, CD45-CD34⁺, and CD7⁺ CD34⁺.Collectively, the results are consistent with approximately 90% of theharvested cells from in vitro cultures having the surface phenotypeCD45⁻CD7⁺ CD34⁺, identical to the atypical phenotype of the primaryleukemia of this patient. This analysis supports the contention thatconditioned medium from cord blood stimulates the in vitro proliferationof primary leukemia cells from patients with T-ALL, rather than simplynormal T cells within the primary T-ALL sample obtained from thepatient.

Consistent with this, it was further shown by flow cytometry, that thecells from this patient in the upper layer supported by the irradiatedcord blood are of leukemia origin and not simply normal T cells from thepatient's blood. Specifically, approximately 80% of the harvested cellsfrom the upper layer of the agar assay, were shown to be CD45⁻CD7⁺phenotype, similar to the atypical phenotype of primary T-ALL from thispatient.

Enhancing activity in cord blood conditioned medium appears to bind to QSepharose at pH 7.5 that was eluted at 500 mM NaCl, and to wheat germagglutinin affinity column that was eluted with 200-300mM-acetyl-D-glucosamine, and, thus, corresponds to an acidicglycoprotein(s). Cytokine IL-15, a potent immunoregulatory cytokine, isalso a T-cell growth factor that can enhance activity ofantigen-specific T cells and lymphokine-activated killer cells. It wasshown that 10 ng/ml of recombinant IL-15 stimulated colony formation ofprimary leukemia by about 80% and neutralizing anti-IL-15 antibody (upto 100 ng/ml) was sufficient to block completely the IL-15 inducedenhancement of leukemia colony formation in the assay. In contrast,similar doses of neutralizing antibody (10 to 100 ng/ml) did not affectthe enhancement attributable to the addition of cord blood conditionedmedium in the assay. These results indicate that IL-15 cannot be theleukemia enhancing activity in the cord blood conditioned medium.Consistent with this, determination by ELISA demonstrated that ourpreparations of cord blood conditioned medium (a total of 22preparations) contains only a negligible amount of IL-15 (<0.03 ng/ml);and this amount is about 100 times less that the ED₅₀ for IL-15biological activities (i.e., 3 ng/ml).

As the in vitro leukemia colony assay using methylcellulose cultureincludes the addition of 100 units/ml of IL-2 and 10 ng/ml of PMA, aninducer of IL-2 receptor, the enhancement of leukemia colony formationby cord blood conditioned medium was unlikely to be due simply to anincrease in IL-2 or IL-2 receptor. Consistent with this, the amount ofIL-2 in the cord blood conditioned medium as determined by ELISA wasfound to be minimal (approximately at 4.3±3.9 units/ml for fourdifferent preparations), as compared to the exogenous IL-2 added in thecultures. Moreover, flow cytometric analysis using anti-IL-2 receptormAbs (M-A251 for IL-2Rα, 3D7 for IL-2RP, and AG184 for IL-2Rγ) indicatesthat cord blood conditioned medium alone has no effect on the expressionof IL-2 receptor.

Mononuclear cells derived from human bone marrow were cultured asdescribed above for cord blood MNCs. The adherent layer, containingmesenchymal stem cells, was isolated as used in both in vitro and invivo studies as described above. Stem cells derived from bone marrowwere found to be effective pre-conditioning agents in the in vivo modelof human leukemia and to enhance leukemic colony formation in vitro.

As set forth above, an animal model of the present invention is suitablemodel of human leukemia. Thus, the model has a variety of uses. One suchuse is the screening of putative anti-cancer (e.g., anti-leukemic)agents. Such a putative agent is administered to the animal model andthe course of leukemia followed over time. Agents can be administeredaccording to any protocol. In addition, the agent can be administeredeither before or after injection of the primary leukocytes.

1. A process for making an in vivo model of human leukemia comprising:a) pre-conditioning an immunodeficient rodent by administering to therodent a sub-lethal dose of irradiation and injecting the rodent with aneffective pre-conditioning amount of human hematopoietic stem cells; b)maintaining the rodent from step (a) for from about 3 to about 12 days;c) injecting the rodent from step (b) with an effective engraftingamount of primary human leukemia cells; and d) allowing the primaryhuman leukemia cells to engraft in the rodent to produce the in vivomodel of human leukemia.
 2. The process of claim 1 wherein theimmunodeficient rodent is an immunodeficient mouse.
 3. The process ofclaim 2 wherein the immunodeficient mouse is a NOD/scid mouse.
 4. Theprocess of claim 1 wherein administering the sub-lethal dose ofirradiation is accomplished by irradiating the rodent with about 350rads of total body gamma radiation.
 5. The process of claim 1 whereinthe effective engrafting amount of primary human leukemia cells is fromabout 10⁶ to about 10⁷ cells.
 6. The process of claim 1 wherein theprimary human leukemia cells are T-cell acute lymphoblastic leukemia(T-ALL) cells.
 7. The process of claim 1 wherein the effectivepre-conditioning amount of human hematopoietic stem cells is from about10⁶ to about 10⁸ cells.
 8. (canceled)
 9. The process of claim 1 whereinthe human hematopoietic stem cells comprise mesenchymal stem cells. 10.The in vivo model of human leukemia produced by the process of claim 1.11. An rodent comprising: human hematopoietic stem cells and engraftedhuman leukemia cells.
 12. The rodent of claim 11 wherein the humanleukemia[-initiating] cells are maintained within the rodent.
 13. Therodent of claim 12 that is a mouse.
 14. The mouse of claim 13 that is aNOD/scid mouse.
 15. The rodent of claim 11 that is irradiated, injectedwith human hematopoietic stem cells, and then injected with humanprimary leukemia cells.
 16. The rodent of claim 11 wherein the engraftedleukemia cells are found in the bone marrow and spleen of the rodent.17. (canceled)
 18. The process of claim 1 wherein the humanhematopoietic stem cells comprise bone marrow stem cells.
 19. Theprocess of claim 1 wherein the human hematopoietic stem cells comprisemononuclear cells.
 20. The process of claim 19 wherein the mononuclearcells comprise human fetal cord blood mononuclear cells.
 21. The processof claim 1 wherein the maintaining step is from 3 to 12 days.
 22. Theprocess of claim 1 wherein the maintaining step is from about 5 to about10 days
 23. The process of claim 1 wherein the maintaining step is from5 to 10 days.